The morphogenesis of biological tubes is central to the development of a wide variety of metazoan structures, from the simplest Cnidarian body plans to the vertebrate respiratory, excretory, and circulatory systems. Although biological tubes form by such distinct processes as the hollowing of single cells and the folding of epithelial sheets, in each case an inner lumen is surrounded by a surface of apical character generated by the polarized movement or growth of vesicles or vacuoles (Hogan, P., et al., Organogenesis: molecular mechanisms of tubulogenesis. Nat. Rev. Genet. 3(7):513–23, 2002; and Lubarsky, M. et al., Tube morphogenesis: making and shaping biological tubes. Cell 112(1):19–28, 2003). A recent model proposes a de novo generation of an apically polarized surface by polarized vesicle targeting and fusion (Lubarsky, et al., supra, 2003). In this model, while developing multicellular tubes target small apical secretory vesicles to a specified region of the plasma membrane where they undergo exocytosis, developing unicellular tubes target to the center of the cell one or more large vacuoles that have originated via a pinocytotic process of invagination (Lubarsky, et al., supra, 2003; see also, J. Folkman and C. Haudenschild, Angiogenesis in vitro. Nature 288(5791):551–6, 1980, and G. E. Davis and C. W. Camarillo, An alpha 2 beta 1 integrin-dependent pinocytic mechanism involving intracellular vacuole formation and coalescence regulates capillary lumen and tube formation in three-dimensional collagen matrix. Exp. Cell Res. 224(1):39–51, 1996).
In general, all developing tubes must achieve the common goals of cell polarization and the establishment and maintenance of tubular architecture, including the precise regulation of tube diameter (G. J. Beitel and M. A. Krasnow, Genetic control of epithelial tube size in the Drosophila tracheal system. Development 127(15):3271–82, 2000). Along a single tubular network, distinct morphogenetic strategies may be used to create tubes of different gauges, and these mechanisms are often conserved across phyla. For example, the process of epithelial tube budding generates both the branches of the mammalian lung and the largest branches of the Drosophila tracheal system (R. J. Metzger and M. A. Krasnow, Genetic control of branching morphogenesis. Science 284(5420):1635–9, 1999). Similarly, the smallest branches of the Drosophila tracheal system are generated via the process of single cell hollowing (G. Manning and M. A. Krasnow, in The Development of Drosophila melanogaster, M. Bate and A. Martinez Arias, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Vol. 1:609–685, 1993), which is also used to generate fine capillaries during mammalian angiogenesis (see, J. Folkman and C. Haudenschild, supra, 1980; G. E. Davis and C. W. Camarillo, supra, 1996). Thus, evolutionarily conserved mechanisms may be expected to function to form tubular structures, and general clues about the morphogenesis and homeostasis of biological tubes may be provided by studying simple tubular networks found in invertebrate model systems.
A simple model of tubular morphogenesis is provided by the Caenorhabditis elegans excretory cell, a single cell that forms the major tubular component of the four cell nematode excretory system (F. K. Nelson, et al., Fine structure of the Caenorhabditis elegans secretory-excretory system. J. Ultrastruct. Res. 82(2):156–71, 1983; F. K. Nelson and D. L. Riddle, Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery. J. Exp. Zool. 231(1):45–56, 1984; and M. Buechner, Tubes and the single C. elegans excretory cell. Trends Cell. Biol. 12(10):479–84, 2002). This cell extends branched tubular processes, termed canals, along the length of the body on the basolateral surface of the epidermis. These processes are seamless yet tunneled by an inner lumen that is closed at its four endings and is presumed to collect fluids and waste, which then empty into the excretory duct. Thus the excretory cell provides a highly tractable model of a seamless, unicellular, fine-gauge tube, such as are found in the secondary branches of Drosophila trachea (G. Manning and M. A. Krasnow, supra, 1993), vertebrate blood capillaries (particularly in the brain)(J. R. Wolff and T. Bar, ‘seamless’ endothelia in brain capillaries during development of the rat's cerebral cortex. Brain Res. 41(1):17–24, 1972), and in other vertebrate organs including the lung (Hogan and Kolodziej, Organogenesis: molecular mechanisms of tubulogenesis. Nat. Rev. Genet. 3(7):513–23, 2002; Lubarsky and Krasnow, supra, 2003).
The process of tubulogenesis has been partially elucidated by the identification of a number of excretory canal mutants (the so-called “exc mutants”) that exhibit characteristic defects in the ability of the canals to form a tubule or regulate the diameter of the excretory cell lumen. (M. Buechner, Tubes and the single C. elegans excretory cell, Trends Cell Biol. 12(10):479–484, 2002; M. Buechner, et al., Cystic Canal Mutants in Caenorhabditis elegans are Defective in the Apical Membrane Domain of the Renal (Excretory) Cell. Dev. Biol. 214:227–241, 1999). All of the twelve exc mutants identified thus far (namely, exc-1, exc-2, exc-3, exc-4, exc-5, exc-6, exc-7, exc-8, exc-9, let-4, let-653, and sma-1) display cyst formation in the excretory canal of C. elegans, although each mutant presents a specific and distinguishing canal morphology, as well as a characteristic variation in cyst size, shape and position. (Buechner, et al., supra, Dev. Biol. 214:227–241, 1999). The exc-4 mutant genotype presents with severe regional enlargements of the excretory canal's interior lumen, marked further with partial septa, which often act to partially or completely occlude the excretory channel. (Buechner, et al., supra, Dev. Biol. 214:227–241, 1999). Further, there is a variable thickness or even frequent absence of the channel cytoskeleton, together with an uneven distribution of the channel canaliculi (thin membranous collecting channels, closed at their distal ends, which feed into lumen of each excretory canal) and lumenal glycocalyx. (Buechner, et al., supra, Dev. Biol. 214:227–241, 1999).
Until the present invention, there has been no disclosure implicating a role in tubulogenesis for any member of the CLIC family of chloride intracellular channel proteins (hereinafter, “CLICs”). CLICs are small proteins that have the unusual property of translocating from a globular cystic form to an integral membrane form (Harrop, et al., Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution. J. Biol. Chem. 276:44993–5000, 2001), where the integral membrane form is associated with chloride channel activity (Ashley, et al., Challenging accepted ion channel biology: p64 and the CLIC family of putative intracellular anion channel proteins. Mol. Membr. Biol. 20:1–11, 2003; Landry, et al., Purification and Reconstitution of Chloride Channels from Kidney and Trachea. Science 244:1469–72, 1989; Li and Weinman, Chloride channels and hepatocellular function: prospects for molecular identification. Annu. Rev. Physiol. 64:609–633, 2002; and Jentsch, et al., Physiol. Rev. Molecular structure and physiological function of chloride channels. 82(2):503–568, 2002). While work in cultured cell systems has indicated CLICs serve roles in a wide variety of diverse processes, such as cell motility (Ronnov-Jessen, Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-betal-mediated conversion of fibroblasts to myofibroblasts. Am. J. Pathol. 161:471–480, 2002), vesicle acidification (Tamir, et al., Secretogogue-induced gating of chloride channels in the secretory vesicles of parafollicular cells. Endocrinology 135(5):2045–2057, 1994), electroneutral acid secretion (Schlesinger, et al., Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption. J. Biol. Chem. 272(30):18636–18643, 1997), cell cycle progression (Valenzuela, et al., The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle. J. Physiol. 529(3):541–552, 2000) and apoptosis (Fernandez-Salas, et al., p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein. J. Biol. Chem. 274:36488–36497, 1999; Fernandez-Salas, et al., mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53. Mol. Cell Biol. 22:3610–3620, 2002), the exact in vivo function role of CLICs has been lacking due to the absence of animal models.
Matthew Buechner, David Hall and Edward Hedgecock first disclosed the existence and phenotype of the C. elegans exc-4 mutant in “Exc Mutations Affect Apical Cytoskeleton” (Early 1995 International Worm Meeting, Abstract 320, 1995) (hereinafter, “Buechner I”). In Buechner I, it is noted that electron microscopy and wheat germ agglutinin staining of mutants defective in excretory canal structure reveals four classes of defects at the apical surface of the excretory cell. One class of nematode mutants, comprising mutations in the exc-1, exc-2, exc-4, exc-5, exc-9, let-4 and let-653 gene loci, exhibit lumena that swell into large cysts coincident with the separation of the apical membrane from its cytoplasmic coat.
The exc-4 mutant phenotype was further characterized by Matthew Buechner, David Hall, Harshida Bhatt and Edward Hedgecock in “Cystic Canal Mutants in Caenorhabditis elegans Are Defective in the Apical Membrane Domain of the Renal Excretory Cell” (Developmental Biology. 214:227–241, 1999) (hereinafter, “Buechner II”). In Buechner II, nematodes were mutagenized to yield 12 different excretory canal mutant phenotypes, wherein each phenotype was associated with mutation in one of the following excretory canal gene loci, to wit, the exc-1, exc-2, exc-3, exc-4, exc-5, exc-6, exc-7, exc-8, exc-9, let-4, let-653 and sma-1 gene loci. Further, three separate mutant alleles of exc-4 were identified, wherein the common phenotype was characterized by a widened lumen, a truncated excretory canal ending well short of the wild-type excretory canal phenotype, and a specific uniform cyst size, shape and position. Finally, using two factor and three factor tests, together with complementation studies, the exc-4 gene locus was roughly mapped as being between the eDf7 and unc54 markers on chromosome I.
Buechner I and Buechner II disclose the existence and characterization of an exc-4 mutant phenotype. Buechner II further identifies three separate mutant alleles of exc-4 (namely, rh133, n561, and n2400), and roughly maps the exc-4 locus to a position in between the unc-54 and eDf7 markers. However, neither of Buechner I or II disclose, suggest or enable a determination of the coding sequence for the exc-4 nucleic acid. Nor does either reference disclose any characterization of the EXC-4 protein. Further, Buechner I and II do not disclose, suggest or enable any method of using C. elegans as an animal model to examine the in vivo function of the CLIC family of chloride intracellular channel proteins. In fact, Buechner II teaches away from a determination that the exc-4 gene is a CLIC orthologue, since it speculates that all of the disclosed excretory canal genes code for functionally related proteins. However, until the present invention, none of the excretory canal genes had been identified as coding for a chloride intracellular channel protein. (See, by way of example, Jones and Baillie, Characterization of the let-653 gene in C. elegans. Mol. Gen. Genet. 248:719–726, 1995 (let-653 encodes a mucin); McKeown, et al., sma-1 encodes a βH-spectrin homolog required for C. elegans morphogenesis. Dev. 125:2087–2098, 1998 (sma-1 encodes the βH-spectrin protein); Fujita, et al., The role of the ELAV homologue EXC-7 in the development of the Caenorhabditis elegans excretory canals. Dev Biol. 256(2):290–301, 2003) (exc-7 encodes a nematode homologue to the neural RNA-binding protein ELAV); and Suzuki, et al., A putative GDP-GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans. EMBO Rep. 2:530–535, 2001) (exc-5 encodes a protein homologous to guanine nucleotide exchange factors)). Finally, neither reference discloses, suggests or enables any method of using screens modulating exc-4 expression (or EXC-4 function) to identify putative agents that inhibit CLIC expression, function or activity.
Various screening methods using C. elegans are identified by Zwaal, et al., in U.S. Pat. No. 6,465,715, entitled “Expression of DNA or proteins in C. elegans”, issued on Oct. 15, 2002 (hereinafter “Zwaal”). Specifically, Zwaal discloses methods for identifying compounds that have an affect on the morphology of the excretory canal, wherein each method includes the expression of a transgene (either a reporter gene and/or a coding sequence for the tested compound) under the control of an excretory canal specific promoter. Using the method of Zwaal, the transgenic C. elegans is first contacted with a candidate compound suspected of being a modulator of the development of the excretory canal of C. elegans, where the transgenic C. elegans comprises any one of a disclosed number of excretory canal specific promoters operatively linked to a reporter gene. A transgenic C. elegans that exhibits an altered excretory phenotype as a result of exposure to the candidate compound is identified, and it is determined whether the compound is a modulator of the development of the excretory canal of C. elegans. 
Zwaal does not disclose, however, the coding sequence of the exc-4 nucleic acid. Nor does Zwaal disclose any characterization of the EXC-4 protein. Further, Zwaal does not disclose, suggest or enable any method of using C. elegans as an animal model to examine the in vivo function of the CLIC family of chloride intracellular channel proteins. Finally, Zwaal does not disclose, suggest or enable any method of using screens modulating exc-4 expression (or EXC-4 function) to identify putative agents that inhibit CLIC expression, function or activity.
Accordingly, in light of the foregoing, there exists a need for an isolated nucleic acid encoding the exc-4 excretory canal gene of C. elegans. Further, a need exists for an animal model representing the in vivo function of the CLIC family of chloride intracellular channel proteins. Finally, there is a need for a high throughput, genetically tractable screen to identify putative agents that inhibit CLIC expression, function or activity.